CA2205906C - In-vivo diagnostic process by near infrared radiation - Google Patents

Abstract

A novel use is provided herein for certain novel compounds for an in vivo diagnostic method based on near infrared radiation. The novel compounds are water-solub le dyes and their biomolecule adducts. Each such dye or adduct has specific photophysica l and pharmaco-chemical properties, which make them suitable as a contrast medium for fluorescence and transillumination diagnostics in the NIR range.

Description

(a) TITLE OF THE INVEN'CION
IN VIVO DIAGNOSTIC 1?ROCESS BY NEAR INFRARED RADIATIC>N
(b) TECHNICAL FIELD TO WHICH THE INVENTION RELATES
This invention relates to the use of new water-soluble dyes and their biomolecule adducts for an in vivo diagnostic method based on near infrared radiation (NIR
radiation).
Each such dye and adduct has specific photophysical and pharmaco-chemical properties, as a contrast medium for fluorescence and transillumination diagnostics in the NIfR range.
This invention also relates to the new dyes.
(c) BACKGROUND ART
Diagnosability of diseases is very much dependent on obtaining information about the structures, as well as changes, of tissues of the profound layers that are not primarily accessible. In addition to palpating, exposing or puncturing these tissues, such information can be gained using sophisticated imaging methods such as X-raying, magnetic resonance tomography, or ultrasonic diagnosis.
As biological tissue shows a relatively high permeability for long wave light in the range of 650 - 1000 nm, a diagnostician can therefore use a completely different method of tissue imaging. The fact that light in the near infrared range can permeate through several centimetres of tissue is utilized in transillumination imaging. This technique ass yet facilitates diagnosis of inflammations of the paranasal and maxillary sinuses as well as the detection of accumulated fluids or blood in superficial zones of tissue (Beutham, J. , Muller, G., Infrarotdiaphanoskopie, Med. Tech. 1 (1992) 13-17).
Attempts at detecting breast tumours have been unsatisfactory so far (Navarro, G.
A.; Profio, A. E.; Contrast in diaphanography of the breast; Med. Phys. 150 (1988) 181-187; Aspegren, K.; Light Scanning Versus , c Y

Mammography for the Detection of Breast Cancer in Screening and Clinical Practice, Cancer 65 (1990) 1671-77) but there may be better results in the future due to most recent engineering progress (Klingenbeck J.; Laser-Mammography with NIR-Light, Gynakol.-Geburtsh.-Randsch.
33 Suppl.l (1993) 299-300); Benaron D.A.; Optical Imaging reborn with technical advances, Diagwostic Imaging (1994) 69-76) .
In addition to detecting non-absorbed radiation, fluorescence radiation emitted after near infrared light treatment can provide tissue-specific information. This so-called autofluorescence is used to distinguish atherosclerotic and normal tissue (Henry, P. D. et al., Laser-Induced Autofluorescence of Human Arteries, Circ.
Res. 63 (1988) 1053-59).
The main problem of applying near infrared radiation is the extraordinarily wide scattering of the light which permits only a rather blurred image of a clearly contoured object despite. different photophysical properties. The problem increases the greater the distance from the surface is and may be considered the major limiting factor of both transillumination and detection of fluorescence radiation.
Suitable fluorescent dyes that accumulate in diseased tissue (above all, in tumours) and that show a specific absorption and emission behaviour, may contribute towards enhancing the distinction of healthy from diseased tissue. The change caused by absorbing irradiated (scattered) light, or fluorescence induced by exciting radiation, is detected and provides the actual tissue-specific information.
inaziiae.noc r t Examples of using dyes for in-vivo diagnostics in humans are photometric methods of tracing in the blood to determine distribution areas, blood flow, or metabolic and excretory functions, and to visualize transparent structures of the eye (ophthalmology). Preferred dyes for such applications are indocyanine green and fluorescein (Googe, J.M. et al., Intraoperative Fluorescein Angiography; Ophthalmoloav, 100, (1993), 1167-70).
Indocyanine green (Cardiogreen) is used for measuring the liver function, cardiac output and stroke volume, as well as the blood flow through organs and peripheral blood flows (I. Med. x(1993)10-27); in addition they are being tested as contrast media for tumour detection.
Indocyanine green binds up to 1000 to albumin and is mobilized in the liver. Fluorescent quantum efficiency is low in a hydrous environment. Its LD50 (0.84 mmol/kg) is great enough; strong anaphylactic responses may occur.
Indocyanine green is unstable when dissolved and cannot be applied in saline media because precipitation will occur.
Photosensitizers designed for use in photodynamic therapy (PDT) (including haematoporphyrin derivatives, photophrin II, benzoporphyrins, tetraphenyl porphyrins, chlorines, phthalocyanines) were used up to now for localizing and visualizing tumours (Bonnett R.; New photosensitizers for the photodynamic therapy of tumours, SPIE Vol. 2078 (1994)). It is a common disadvantage of the compounds listed that their absorption in the wavelength range of 650 - 1200 nm is only moderate. The phototoxicity required for PDT is disturbing for purely diagnostic purposes. Other patent specifications dealing with these topics are: U S -PS 494529; WO 84 04665, WO 90/10219, n'E-OS 4136769, DE-PS 2910760. __ ID42114&.DOC

, LT. S.-PS 4945239 describes a great number of equipment arrangements for detecting breast cancer using transillumination and mentions the known fluorescein, fluorescamin, and riboflavin as contrast-improving absorption dyes. These dyes share the disadvantage that they absorb in the visible wavelength range of 400-600 nm in which light transmission capacity of tissue is very low.
DE-OS 4136769 describes an apparatus for detecting fluorescence of tissue areas enriched with fluorescent substances. These substances are bacterial chlorophyll and its derivatives, and naphthalocyanines. These structures show absorptions in the range of 700-800 nm at absorbency indices of up to 70000 1 mol-1 cm-1. In addition to their fluorescent properties, the compounds mentioned here are capable of generating singlet oxygen by radiation, thus having a cytotoxic effect (photosensitizers for photodynamic therapy). This photosensitizing activity is highly undesirable for a pure, inactive diagnostic agent.
Furthermore, synthesis of bacterial chlorophyll compounds is expensive and requires much effort as natural products have to be used as parent substances; the naphthalocyanines, however, frequently show a very low photostability. The known compounds of these classes are hardly soluble in water, and synthesizing uniform hydrophilic derivatives is costly.

5 describes an in-vivo method for the fluorescence detection of tumours using the following photosensitizers: haematoporphyrin and its derivative(Hp and HpD), uro- and copro- and protoporphyrin as well as numerous mesosubstituted porphyrins, and dyes such as riboflavin, fluorescein, acridine orange, berberine ID42114H.DOC

sulfate and tetracyclines. The photophysical and pharmacochemical requirements mentioned above are not met by said substances.
5 Folli et al., Cancer Research ~, 2643-2649 (1994), describe a monoclonal antibody connected with a cyanine dye that was used for detecting a tumour implanted subcutaneously. Detection of profounder pathologic processes, however, requires much improved dyes. Higher dye dosages render the use of antibodies as carriers unsuitable in view of the side effects to be expected.
Cyanine dyes and polymethine dyes related to them are also used as photographic layers. Such dyes need not have any luminescent properties. Cyanine dyes that have luminescent (fluorescent) properties have been synthesized for use in fluorescent microscopy and flow cytometry and coupled with biomolecules such as compounds containing iodine acetyl groups as specific labelling reagents for sulfhydryl groups of proteins (Waggoner, A.S. et al.; Cyanine dye Labeling Reagents for Sulfhydryl Groups, Cvtometrv. 10, (1989), 3-10). Proteins are labelled and isolated in this way. More references:
Cytom~trv 12 (1990) 723-30; Anal. Lett. 25 (1992) 415-28;
Bioconjugrate Chem. 4 (1993) 105-11.
DE-OS 39 12 046 by Waggoner, A.S. describes a method for labelling biomolecules using cyanine and related dyes such as merocyanine and styryls that contain at least one sulfonate or sulfonic acid grouping. This specification relates to a single and two-step labelling method in a hydrous environment, with a covalent reaction taking place between the dye and the amine, hydroxy, aldehyde or sulfhydryl group on proteins or other biomolecules.
ID42119&.DDC

DE-OS 3828360 relates to a method for labelling antitumour antibodies, in particular, antibodies specific to melanoma and colonic cancer, using tluorescein and indocyanine green for ophthalmologic purposes. Bonding of indocyanine green to biomolecules is not covalent (dye-antibody combination, mixture).
The known, state-of-the-art methods of in vivo diagnosis using NIR radiation thus show a number of disadvantages that prevented their wide application in medical diagnostics.
Direct use of visible light: or NIR radiation is restricted to superficial body zones, which is due to the widely scattered incident light.
Adding dyes to improve contrast and resolution, however, gives rise to a number of other problems. The dyes should meet the requirements that generally apply to diagnostic pharmaceuticals. As these substances are mostly applied at higher doses and for a longer diagnostic period, they should be low-toxic. In addition, dyes suitable for diagnostic purposes should be well soluble in water and sufficiently stable in chemical and photophysical respect, at least for as long as the diagnostic period lasts.
Stability as regards metabolization in the system is also desirable.
So far, neither dyes nor a suitable method for in vivo diagnosis using NIR
radiation have been available.
(d) DESCRIPTION OF THfE INVENTION
It is therefore an object of a first aspect of this invention to provide an in vivo diagnosis that mitigates substantially overcomes the disadvantages of prior art.
This problem is solved according to the invention by providing a method of in vivo diagnosis using NIR radiation in which compounds of the general Formula I
B~ - (F-~'~'")~n (I) An object of a second aspect of the invention is to provide novel dyes for use in in vivo diagnosis.
One aspect of the present invention provides the use of compounds of the general Formula I

B~ - (F-W")m wherein:
1 represents a number from 0 to 6, n represents a number from 0 to 10 and m represents a number from 1 to 100;
B is a biological detecting unit having a molecular weight of up to 30,000 that bonds to specific cell populations or selectively to receptors, or accumulates in tissues or tumours, or generally stays in the blood, and is further selected from the group consistiang of an amino acid, a peptide, a complementary determining region, an antigen, a hapten, an enzyme substrate, an enzyme cofactor, biotin, a carotenoid, a hormone, a neurohormone, a neurotransmitter, a growth factor, a lymphokin, a lectin, a toxin, a carbohydrate, an oligosaccharide, a polysaccharide, a dextrane, an oligonucleotide made resistant to nucleases and a receptor-bonding pharmaceutical, or is a macromolecule that bonds non-selectively, the macromolecule being selected from the group consisting of polylysine, polyethylene glycol, methoxypolyethylene glycol, polyvinyl alcohol, dextrane, carboxydextrane or a cascade-polymer-like structure that is covalently bonded to F;
F represents a dye showing maximum absorption in the range of 650 to 1200 nm;
W represents a hydrophilic group that improves water-solubility, with the n-octanol-water distribution coefficient of the compound according to Formula I being less than, or equal to, 2.0 for 1 = 0;
or their physiologically-tolerable salts, in an in vivo diagnostic method based on near infrared radiation.
A first variant of this first aspect of the present invention is a use whert~
F within general Formula I represents a cyanine dye of the general Formula IIa R3 R' 4 $
R / x - Y ~ R
+~~'L_r-~2-~3y 4-~5 ~6 Rs ~ N r N ~ Rs i i Rs Rn R'2 Rb (IIa) g wherein:
r represents the numbers 0, 1 or 2, on condition that, for r = 2, the respective fragments L6 and L' that occur in duplicate may be same or different;
L' to L' are same or different, each independently representing a fragment ClH
or CR, where R is a halogen atom, a hydro~;y, a carboxy, an acetoxy, an amino, a nitro, a cyano or a sulphonic acid group; or an alkyl, an alkenyl, a hydroxyalkyl, a carboxyalkyl, an alkoxy, an alkoxycarbonyl, a sulphoalkyl, an alkylamino, a dialkylamino or a halogenalkyl residue containing up to 6 carbon atonns; or an aryl, an alkylaryl, a hydroxyaryl, a carboxyaryl, a sulphoaryl, an arylamino, a diarylamino, a nitroaryl or a halogenaryl residue containing up to 9 carbon atoms; or where R: represents a bond that bonds to another residue R and forms a 4- to 6- member ring together with the interspersed residues L' to L' ; or where R
represents one bond, respectively, at two different positions that are linked via a -CO-fragment;
R3 to R'2 are same or differenr., each independently representing a hydrogen .atom, a residue B or W, as defined above, or an alkyl or alkenyl residue containing up to 6 carbon atoms, or an aryl or aralkyl residue containing up to 9 carbon atoms, the alkyl, alkenyl, aryl or aralkyl residue carrying one r~°sidue W, as defined above, or carrying an additional residue W as defined above which is annealed to each pair of adjacent residues R3 to R'° with due regard for the interspersed C .atoms, or saturated, unsaturated or aromatic 5-to 6-membered rings having zero or one additional residue as defined above;
X and Y are same or different, each independently representing an O, S, Se or Te or a -C(CH3)Z-, -CH=CH- or -CR'3 R'4- fragment, where R'3 and R'4 independently represent a hydrogen atom, a residue B or W, as defined above, or an alkyl or alkenyl residue containing up to 6 carbon atoms or an aryl or aralkyl residue containing up to 9 carbon atoms, the alkyl, alkenyl, aryl or aralkyl residue carrying zero or one additional residue W, as defined above; or represents a squarain dye of the general Formula IIb R
gs a O It~~ yo IIb wherein:
s and t independently represent the numbers 0 or 1, on the condition that s and t do not represent the number I at the same time;
and R3 to R'z, X and Y are as defined above; or represents a styryl dye of the general Formula IIc R
R~,RB

L1= L2 L L --f ~ L6 / N
\Rn ~I' R Ft IIc wherein:
r, L' to L6, R3 to R" and X are as defined above; or represents a merocyanine dye of the general Formula IId 1~

HO R' X
N
l ~2 l3 L -- f ~5- ~6 = G
r N
11 p \Ra IId wherein:
r, L' to L6, R3 to R8, R" and X are as defined above, and G represents an oxy;~en or sulphur atom.
A second variant of this first aspect of the present invention is a use where F within the general Formula I represents a squarain dye of the general Formula IIb R3 OH R'7 R / X Y ,~ R
,CH-CH CH = ~ CH ~ CH CH
Rs ~ N ~ S ~ t N W Rs Rs R" ~ R'2 Rio IIb wherein:
R3 to R'2 are same or different, each independently representing a hydrogen atom, a residue B or W as is defined above, or an alkyl or alkenyl residue containing up to 6 carbon atoms or an aryl or aralkyl residue containing up to 9 carbon atoms, the alkyl, alken.yl, aryl or aralkyl residue carrying zero or an additional residue W to which each pair of adjacent residues R3 to R'° are annealed, with due regard for the interspersed atoms; or 5- to 6-member rings that may be sal:urated, unsaturated or aromatic, and carrying zero or one residue R, wherein R is a halogen atom, a hydroxy, a carboxy, an acetoxy, an amino, a vitro, a cyano or a sulphonic acid group, or an alkyl, an alkenyl, a hydroxyalhyl, a carboxyalkyl, an alkoxy, an alk:oxycarbonyl, a sulphoalkyl, an alkylamino, a dialkylamino or a halogenalkyl residue containing up to 6 carbon atoms; or an aryl, an alkylaryl, a hydroxyaryl, a carboxyaryl, a sulphoaryl, an arylamino, a diarylamino, a nitroaryl or a halogenaryl residue containiing up to 9 carbon atoms;
s and t independently represent the numbers 0 to 1, on the condition that s and t do not represent the number 1 at the same time, X and Y are same or different, each independently representing an O, S, S~e or Te or a -C(CH3)2-, -CH=CH- or -C'.R'3R''~- fragment, wherein R'3 and R'~ independently represent a hydrogen atom, a residue B as defined in claim 2, or W as defined herein below, or an alkyl or alkenyl residue containing up to 6 carbon atoms, or an aryl or aralkyl residue containing up to 9 carbon atoms, the alkyl, alkenyl, aryl or aralkyl residues having zero or one residue W;
W represents a hydrophilic group that improves water-solubility, with the n-octanol-water distribution coefficient of the compound according to Formula I being less than, or equal to, 2.0 for 1=0;
as well as their physiologically tolerable salts.
A third variant of this first aspect of the present invention is a use where W
within the general Formula I is selected from the group consisting of (i) a carboxy or sulphonic acid group; (ii) a carboxyalkyl group; (iii) an alkoxycarbonyl or alkoxyox~oalkyl group containing up to 12 carbon sulphonic atoms; (iv) a residue of the general Formulae III
-(CHZ);, -O-Z or (-CHZ-CHZ-O)d-Z (III) wherein:
a represents the numbers 0 to 6;
Z represents a hydrogen atom or an alkyl residue containing 3 to 6 C atoms that includes 2 to n-1 hydroxy groups, witlh n being the number of C atoms, or an aryl or aralkyl residue containing 6 to 10 C atoms and having 2 to 4 additional hydroxy groups, or an alkyl residue containing 1 to 6 C atoms and having 1 to 3 additional carboxy groups, or an aryl residue containing 6 to 9 C atoms and having 1 to 3 additional carboxyl groups, or an aralkyl residue or a nitroaryl residue or a nitroaralkyl residue each containing 6 to 15 C
atoms, or a sulphoalkyl residue containing 2 to 4 C atoms having 1 to 3 additional carboxy groups, or represents a residue of the general Formulae IIIa or IIIb n -c _ _ ~~ ~-cocu~
C ~N~N~--~N~-COOH
COOH COOH
~COOH ~COCH ~COCH
IIIb;
IIIa or represents a residue of the general Formula IIIc -(CHz)~ -CO)~-NR'-(CH~)s-(NH-CO)4-RZ
(IIIc) wherein:
o and s independently represent the numbers, 0, 1, 2, 3, 4, 5 or 6;
p and q independently represent 0 to 1;
R' and RZ independently represent a residue Z, as defined above, except the substituents of the general Formulae IIIa and Illb, or independently represent a residue of the general Formulae IIId or IIIe - CH2 - CHi \ COOH
- \N~N/--COOH
COON ~ COOH ~ COOH ~ COOH ~ C00H
IIIe;
IIId or represents a residue of the general Formula IIIc, as defined above.

A fourth variant of this first aspect of the present invention is a use where F within the general Formula I represents a cyanine dye of the general Formula V
R2o Rza Y
+~ (:H= Q- Ct-~< ( V ;
N N ~ Rzs I
C~R2s R2s ~~ R27 where Q is a fragment selected from the group consisting of R3o R3o R3o I I I
= CH- C= CH- _~ CH- CI-~ C- CF-~ CH- = CH- C= C- C= Cit--ccH2) b~

and OFt~~
where R3° represents a hydrogen atom, a hydroxy group, a carboxy group, an alkoxy residue containing 1 to 4 carbon atoms or a chlorine atom, b is an integer 2 or 3, R3' represents a hydrogen atom or an alkyl residue containing 1 to 4 carbon atoms, X and Y independently represent an -O-, -S-, or a -CH=CH- fragment or -C(CHZR3z)(CH, R33)-fragment, R'° to RZ~, R32 and R33 independently represent a hydrogen atom, a hydroxy group, a carboxy acid residue, a sulphonic acid residue or a carboxyalkyl residue, an alkoxycarbonyl residue or an a.lkoxyoxoalkyl residue, each containing up to 10 C atoms, or a sulphoalkyl residue containing up to 4 C atoms, or a non-selectively bonding macromolecule or a residue of the general Formula VI

-(O)~ -(CHZ)~, -CO-NR~'' -(CHZ), -(NH-CO)q -R3s (VI) on the condition that, where: X and Y are O, S, -CH =CH- or -C(CH3)Z-, apt least one of the residues Rz° to RZ'' corresponds to a non-selectively bonding macromoleculle or a compound of the general Formula VI, above where:
o and s equal 0 or independently represent an integer between 1 and 6;
q and v independently represent 0 or 1;
R34 represents a hydrogen atom or a methyl residue;
R3s represents an alkyl residue containing 3 to 6 C atoms and comprising 2 to n-1 hydroxy groups, with n being the number of C atoms, or an alkyl residue containing 1 to 6 C atoms that carries 1 to 3 additional carboxy groups, an aryl residue containing 6 to 9 C atoms or arylalkyl residue containing 7 to 15 C atoms, or a residue of the general Formulae IIId or IIIe COOH - CHi N/'~N~N~ ~N~N,-COOH
COON ~ COOH ~ COOH ~ COOH ~ COOH
IIId IIIe, on the condition that q is l, or a non-selectively bonding macromolecule;
Rz° and RZ', RZ' and RZZ, Rz2 and R23, R24 and R's, Rzs and R''', RZG
and RZ7, together with the interspersed carbon atoms, form a 5- or 6-member annealed aromatic or saturated ring;
as well as their physiologically tolerable salts.
A fifth variant of this first aspect of the present invention is a use where the dye comprises: 5-[2-[( 1,2-dicarboxyethyl)amino]-2-oxoethyl]-2-[7-[5-[2-[( 1,2-dicarboxyethyl) amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-(4-sulphobutyl)-2H-indol-:?-yliden]-I ,3,5-IS
heptatrienyl]-3,3-dimethyl-1-(4-sulphobutyl)-3H-indolium, inner salt, potassium hydrogen salt.
A sixth variant of this first aspect of the present invention is a use where the dye comprises: 2-[7-[5-[2-[(11-carboxy-2-oxo-1,4,7,10-tetraaza-4,7,10-tri(carboxyrnethyl)-1-undecyl)amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-ethyl-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulphobutyl)-3H-indolium, inner salt.
A seventh variant of this first aspect of the present invention is a use where the dye comprises:2-[7-[ 1,3-dihydro-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)-ami no]-2-oxoethyl]-1-(4-sulphobutyl)-2I-I-indol-2-yliden]-I ,3,5-heptatrienyl]-3,3-dimethyl-5-[2-(methoxypolyoxyethylene)amino]-2-oxoethyl]-1-(4-sulphobutyl)-3H-indolium, sodium salt.
An eighth variant of this first aspect of the present invention is a use where the dye comprises: 2-[7-[1,3-dihydro-3,:3-dimethyl-1-(4-sulphobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxypolyoxyethylene)aminocarbonyl-1--(4-sulphobutyl)-3H-indolium, sodium salt.
A ninth variant of this first aspect of the present invention is a use where the dye comprises: 3-(3-carboxypropyl)-2-[7-[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulphobutyl)-2H-indol-2-yliden]-I ,3,5-heptatrienyl]-3-methyl-I-(4-sulphobutyl )-3H-indolium, sodium salt.
A tenth variant of this first aspect of the present invention is a use where the dye comprises: 2-[[3-[[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulphobutyl)2H-indol-2-yliden]methyl]-2-hydroxy-4-oxo-2-cyclobuten-1-yliden]methyl]-1,1-dimethyl-3-ethyl-1H-2-benz(e)indolium, inner salt.
An eleventh variant of this first aspect of the present invention is a use where the dye comprises: 2-[7-[1,3-dihydro-5--[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3 , 3-dimethyl-1-(4-sulphobutyl)-~2H-indol-2-yl iden]-1, 3 , 5-heptatrienyl]-5-[2-[(2, , 3-dihydroxypropyl)amino]-2-oxotahyl]-3,3-dimethyl-1-(4-sulphobutyl)-3H-indolium, sodium salt.
A second aspect of the present invention provides an agent for in vivo diagnosis comprising a compound of the general Formula I

wherein:
1 represents a number from 0 to 6, n represents a number from 0 to 10 and m represents a number from 1 to 100;
B is a biological detecting unit having a molecular weight of up to 30,000 that bonds to specific cell populations or selectively to receptors, or accumulates in tissues or tumours, or generally stays in the blood, and is further selected from the group consisting of an amino acid, a peptide, a complementary determining region, an antigen, a hapten, an enzyme substrate, an enzyme cofactor, biotin, a carotenc»d, a hormone, a neurohormone, a neurotransmitter, a growth factor, a lyrnphokin, a lectin, a toxin, a carbohydrate, an oligosaccharide, a polysacchari~Je, a dextrane, an oligonucleotide made resistant to nucleases and a receptor-bonding pharmaceutical, or is a macromolecule that bonds non-selectively, the macromolecule being selected from the group consisting o~f polylysine, polyethylene glycol, methoxypolyethylene glycol, polyvinyl alcohol, dextrane, carboxydextrane or a cascade-polymer-like structure that is covalently bonded to F;
F represents a dye showing maximum absorption in the range of 650 to 1200 nm;
W represents a hydrophilic group that improves water-solubility, with the n-octanol-water distribution coefficient of the compound according to Formula I being less than, or equal to, 2.0 for 1 = 0;
or their physiologically-tolerable salts, in an in vivo diagnostic method based on near infrared radiation, together with at least one of physiologically-acceptable adjuvants, substrates and diluents.
A first variant of the second aspect of the present invention comprises a compound of general Formula I wherein F within that Formula represents a cyanine dye of the general Formula IIa R3 R' R / X -. Y w R

+,~-L' ==LZ-L3=L4-LS
Rs \ N r N i. Rs i i Rs Rn R~2 Rya (IIa) wherein:
r represents the numbers 0, 1 or 2, on condition that, for r = 2, the respective fragments L6 and L' that occur in duplicate may be same or different; L' to L' are same or different, each independently representing a fragment CH or CR, where R is a halogen atom, a hydroxy, a carboxy, an acetoxy, an amino, a nitro, a cyano or a sulphonic acid group; or an alkyl, an alkenyl, a hydrox;yalkyl, a carboxyalkyl, an alkoxy, an alkoxycarbonyl, a sulphoalkyl, an alkylamino, a dialkylamino or a halogenalkyl residue containing up to 6 carbon atoms; or an aryl, an alkylaryl, a hydroxyaryl, a carboxyaryl, a sulphoaryl, an arylamino, a diarylamino, a niitroaryl or a halogenaryl residue containing up to 9 carbon atoms; or where R represents a bond that bonds to another residue R and forms a 4- to 6-member ring together with the interspersed residues L' to L' ; or where R
represents one bond, respectively, at two different positions that are linked via a -CO-fragnnent; R3 to R'z are same or different, each independently representing a hydrogen atom, a revsidue B or W, as defined above, or an alkyl or alkenyl residue containing up to 6 carbon atoms, or an aryl or aralkyl residue containing up to G carbon atoms, the alkyl, alkenyl, aryl or aralkyl residue carrying one residue W, as defined above, or carrying an additional residue W as defined above which is annealed to each pair of adjacent residues R3 to R'° with due regard for the interspersed C atoms, or saturated, unsaturated or aromatic 5- to 6-nnembered rings having zero or one additional residue as defined above; X and Y are same or different, each independently representing an O, S, Se or Te or a -C(CH3)Z-, -CH=CFI- or -CR'3 R'4-fragment, where R'3 and R'4 independently represent a hydrogen atom, a residue B or W, as defined above, or an alkyl or alkenyl residue containing up to 6 carbon atoms or an aryl or aralkyl residue containing up to ~ carbon atoms, the alkyl, alkenyl, aryl or aralkyl residue carrying zero or one additional residue W, as defined above; or represents a squarain dye of the general Formula IIb Rte ato IIb wherein:
s and t independently represent the numbers 0 or 1, on the condition that s and t do not represent the number 1 at the same time; and R~ to R'2, X and Y are as defined above; or represents a styryl dye of the general Formula IIc R
R'.R°
R°
L~= L? L l --f L L6 / - N
\R»
N' R R
IIC
wherein: r, L' to L6, R3 to R' ' and X are as defined above; or represents a me~rocyanine dye of the general Formula IId HO R' N
L ~= L2- L3= L -f LS= L6 ~ = G
r N. N
11 0 \Re R
IId wherein:

r, L' to L6, R3 to R8, R" and X are as defined above, and G represents an oxyf;en or sulphur atom, together with at l~°ast one of physiologically-acceptable adjuvants, substrates and diluents.
A second variant of the second aspect of the present invention comprises a compound of general Formula I wherein F within that Formula represents a squarain dye of the general Formula IIb R~
Rsi w "N N- ~ ~R9 a p R«
IIb wherein:
R3 to R'Z are same or different, each independently representing a hydrogen atom, a residue B or W as is defined above, or an alkyl or alkenyl residue containing up to 6 carbon atoms or an aryl or aralkyl residue containing up to 9 carbon atoms, the alkyl, alken,yl, aryl or aralkyl residue carrying zero or an additional residue W to which each pair of adjacent residues R3 to R'° are annealed.. with due regard for the interspersed atoms; or 5- to 6-member rings that may be saturated, unsaturated or aromatic, and carrying :zero or one residue R, wherein R is a halo~;en atom, a hydroxy, a carboxy, an acetoxy, an amino, a vitro, a cyano or a sulphonic acrid group, or an alkyl, an alkenyl, a hydroxyalkyl, a carboxyalkyl, an alkoxy, an alf;oxycarbonyl, a sulphoalkyl, an alkylamino, a dialkylamino or a halogenalkyl residue containing up to 6 carbon atoms; or an aryl, an alkylaryl, a hydroxyaryl, a carboxyaryl, a sulphoaryl, an arylamino, a diarylamino, a nitroaryl or a halogenaryl residue containing up to 9 carbon atoms; s and t independently represent the numbers 0 to 1, on the condition that s and t do not represent the number 1 at the same time, X and Y are same or different, each independently representing an O, S, Se or Te or a -C(CH3)2-, -CH=CH- or -CF;'3R'~- fragment, wherein R'3 and R'4 independently represent a hydrogen atom, a residue B as defined in claim 2, or W as defined herein below, or an alkyl or alkenyl residue containing up to 6 carbon atoms, or an aryl or aralkyl residue containing up to 9 carbon atoms, the alkyl, alkenyl, aryl or aralkyl residue having zero or one residue W; W represents a hydrophilic group that improves water-solubility, with the n-octanol-water distribution coefficient of the compound according to Formula I
being less than, or equal to. 2.0 for l=0; as well as their physiologically tolerable salts, together with at least one oi; physiologically-acceptable adjuvants, substrates, and diluents.
A third variant of the second aspect of the present invention comprises a compound of general Formula I wherein W within that general Formula I represents is selected from the group consisting of (i) a carboxy or sulphonic acid group; (ii) a carbor:yalkyl group;
(iii) an alkoxycarbonyl or alkoxyoxoalkyl group containing up to 12 carbon sulphonic atoms; (iv) a residue of the general Formulae III
-(CHZ)a -O-Z or (-CHZ-CHZ-O)a-Z (III) wherein:
a represents the numbers 0 to 6; Z represents a hydrogen atom or an alkyl residue containing 3 to 6 C atoms that includes 2 to n-1 hydroxy groups, with n being the number of C atoms, or an aryl or aralkyl residue containing 6 to 10 C atoms and having 2 to 4 additional hydroxy groups, or an alkyl residue containing 1 to 6 C atoms .and having 1 to 3 additional carboxy groups, or an aryl residue containing 6 to 9 C atoms and having 1 to 3 additional carboxyl groups., or an aralkyl residue or a nitroaryl residue or a nitroaralkyl residue each containing 6 to 15 C atoms, or a sulphoalkyl residue containiing 2 to 4 C
atoms having 1 to 3 additional carboxy groups, or represents a residue of the general Formulae IIIa or IIIb -o-~k - c - cH~ w-~ ~- CooH
N~N~N~ C~ N N
~COCiH ~COOH
~COOH ~COCH ~C(xH
IIIa IIIb;

or represents a residue of thf: general Formula IIIc -(CHZ)~, -CO)~-NR'-(CHZ)S-(NH-CO)y-RZ
(IIIc) wherein: o and s independently represent the numbers, 0, I , 2, 3, 4, 5 or 6~;
p and q independently represent 0 to I ; R' and Rz independently represent a residue Z, as defined above, except the substituents of the general Formulae IIIa and IIIb, or independently represent a residue of the general Formulae IIId or IIIe - CHz ~ ~ COON
N N N N NI
COOH ~ COOH ~ COOH
COOH \ COOH
IIIe;
IIId or represents a residue of the general Formula IIIc, as defined above"
togeaher with at least one of physiologically-acceptable adjuvants, substrates and diluents.
A fourth variant of the: second aspect of the present invention comprises a compound of general Formula I wherein F within that Formula represents a cyanine dye of the general Formula V
Rza Rza R2~ Y \ R25 I +~- CH= Q- CI-i=<
R2z \ N N / zs V
R
Rz3 CI~I~ze Rzs ~~ Rz7 , where Q is a fragment selected from the group consisting of R3o R3o _ R3o I i I
= CH- C= CH- = CH- CH= C- CH= CH- = CH- C= C- ~ CH-~-~cH2~ b~

and OR~~
where R3° represents a hydrogen atom, a hydroxy group, a carboxy group, an alkoxy residue containing 1 to 4 carbon atoms or a chlorine atom, b is an integer 2 or 3, R3' represents a hydrogen atom or an alkyl residue containing 1 to 4 carbon atoms, X and Y
independently represent an -O-, -S-, or a -CH=CH- fragment or -C(CHZR3')(CH, R33)-fragment, R'° to R"', R3' and R33 independently represent a hydrogen atom, a hydroxy group, a carboxy acid residue, a sulphonic acid residue or a carboxyalkyl residue, an alkoxycarbonyl residue or an alkoxyoxoalkyl residue, each containing up to 10 C atoms, or a sulphoalkyl residue containing up to 4 C atoms, or a non-selectively bonding macromolecule or a residue of the general Formula VI
-(O)~ -(C:HZ)~ -CO-NR34 -(CHZ)5 -(NH-CO)q -R3s (VI) on the condition that, where X and Y are O, S, -CH=CH- or -C(CH3)2-, at least one of the residues RZ° to R29 corresponds to a non-selectively bonding macromolecule or a compound of the general Formula VI, above where: o and s equal 0 or independently represent an integer between 1 and 6; q and v independently represent 0 or 1; R34 represents a hydrogen atom or a methyl residue; R35 represents an alkyl residue containing 3 to 6 C
atoms and comprising 2 to n-1 hydroxy groups, with n being the number of C atoms, or an alkyl residue containing 1 to 6 C atoms that carries 1 to 3 additional carboxy groups, an aryl residue containing 6 to 9 C atoms or arylalkyl residue containing 7 to 15 C
atoms, or a residue of the general Formulae IIId or IIIe CAN ~ N,--~ N /- COOH C N2~ N,- COOH
COON ~ COOH ~ COOH ~ COOH ~ COOH
IIId IIIe, on the condition that q is 1, or a non-selectively bonding macromolecule;
RZ° and R'', R'' and Rz2, Rzz and R23, R'-4 and Rz'', Rz5 and R''', R~'' and R'', together with the interspersed carbon atoms, form a 5- or 6-member annealed aromatic or saturated ring; as well as their physiologically tolerable salts, together with at least one of physiologically-acceptable adjuvants, substrates and diluent .
A fifth variant of the second aspect of the present invention comprises the cyanine dye 5-[2-[(1,2-dicarboxyethyl)amino)-2-oxoethyl]-2-[7-[5-[2-[(1,2-dicarboxyethyl) amino]-2-oxoethyl]-1, 3-dihydro--3 , 3-dimethyl-1-(4-sulphobuty 1)-2H-indol-2-yl iden]-1, 3 , 5-heptatrienyl]-3,3-dimethyl-1-(4-sulphobutyl)-3H-indolium, inner salt, potassiurn hydrogen salt together with at least one of physiologically acceptable adjuvants, substrates, and diluents.
A sixth variant of the second aspect of the present invention comprises the cyanine dye 2-[7-[5-[2-((11-carboxy-2-oxo-1,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-undecyl)amino]-2-oxoethyl]-1, 3~-dihydro-3,3-dimethyl-1-ethyl-2H-indol-2-ylidew]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulphobutyl)-3H-indolium, inner salt together with at least one of physiologically acceptable adjuvants, substrates, and diluents.
A seventh variant of the second aspect of the present invention comprises. the cyanine dye 2-[7-[1,3-dihydro-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)-amino]-2-oxoethyl]-1-(4-sulphobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)amino]-2-oxoethyl]-1-(4-sulphobutyl)-3H-indolium, sodium salt together with at least one of physiologically-acceptable adjuvants, substrates, and diluents.

An eighth variant of the second aspect of the present invention comprises the cyanine dye 2-[7-[1,3-dihydro-3,3-dimethyl-1-(4-sulphobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxypolyoxy-ethylene)aminocarbonyl-1-(4-sulphobutyl)-3H-indolium, sodium salt together with at least one of physiologically-acceptable adjuvants, substrates, and diluents.
A ninth variant of the second aspect of the present invention comprises the cyanine dye 3-(3-carboxypropyl)-2-[7-[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-I-(4-sulphobutyl)-2H-indol-2-ylidc~n]-1, 3,5-heptatrienyl]-3-methyl-1-(4-sulphobutyl)-3H-indolium, sodium salt together with at least one of physiologically acceptable adjuvants, substrates, and diluents.
A tenth variant of the secomd aspect of the present invention comprises the cyanine dye 2-[[3-[[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulphobutyl)2H-ind~ol-2-yliden]
methyl]-2-hydroxy-4-oxo-2-cyclobuten-1-yliden]methyl]-I ,1-dimethyl-3-ethyl-1 H-2-benz(e) indolium, inner salt together wivth at least one of physiologically acceptable adjuvants, substrates, and diluents.
An eleventh variant of the second aspect of the present invention comprises the cyanine dye 2-[7-[1,3-dihydro-_'~-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-~3,3-dimethyl-1-(4-sulphobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-5-[2-[(2, 3-dihydroxypropyl)amino]-2-oxoethyl]-:3,3-dimethyl-1-(4-sulphobutyl)-3H-indolimm, sodium salt together with at least one oi= physiologically-acceptable adjuvants, substrates, and diluents.
(e) GENERALIZED DESCIftIPTION OF THE INVENTION
The compounds whose use constitutes broad aspects of the present invention are those which absorb and fluoresce in the wavelength range of 650 to 1200 nm, have absorption coefficients of 100,000 1 mol-~ cirri' and more and, where fluorescence is desirable, have a fluorescence quantum efficiency greater than 5 % , are sufficiently water-solublie, tolerable and stable in vitro and in vivo as well as photostable. They are discharged as completely as possible in as short a time as possible. The compounds whose use constitutes broad aspects of the present invention are synthesized easily and at a favourable price in only few reaction steps from parent materials that are available on the market.
In the use according to aspects of the present invention in in vivo diagnosis, one or several substances of the general Formula I (as defined hereinabove) is, or are, administered to the tissues, for example, by intravenous injection, then they .are irradiated with light from the visible to the near infrared range of 650 to 1200 nm.
Radiation that is not absorbed and fluorescence radiation are recorded separately or simultaneously, or against each other with a delay. A synthetic image is generated from the data.
obtained.
Fluorescent images can'be recorded using various methods. Preferred are those methods where the tissue is irradiated extensively, fluorescence information is visualized in local resolution by a CCD cannera, or where the tissue sectors to be imaged are scanned by a light ray concentrated in a fibre optical waveguide and signals which are obtained are converted into an image by computing. The light is beamed-in in the narrow--band range at wavelengths which are close to the maximum absorption or at fluorescence-exciting wavelengths of the compounds of other aspects of the present invention.
Radiation that is not absorbed can be recorded as described, and signals so-obtained be processed.
Irradiation angle and they angle of observation can be selected from case to case to meet anatomic and optimum contrast requirements. The sensitivity of the use of aspects of the present invention involving the use of these novel compounds of other aspects of the present invention may be improved by subtracting the images prior to, and after, administering the dye. Evaluating the time curve of dye-related changes may reveal useful additional information for the diagnosis.
The measurement methods used are known to a person skilled in the art. The expert will also know what equipment parameters should be set to obtain optimum recording and evaluation conditions at given absorption or fluorescence wavelengths of the dyes of the general Formula I of other aspects of the present invention which are used according to aspects of the present invention.
The compounds of the general Formula I of other aspects of the present invention whose use constitutes methods of aspects of the present invention cover a wide range of exciting and emission wavelengths due to the variable structure of the dye system F (as described above). It is possible to gain products with exciting wavelengths that correspond to a specific source of excitation, e. g., at the diode laser unit, and are therefore adapted to a given measuring system or equipment component.
The techniques described even permit localization of small objects having a volume of only a few mm' at the profounder layers of tissue or in non-transparent body fluids. Due to light scattering and the limited resolution it entails, it is still difficult to determine the exact shape and size of such objects hut this is not required to solve some important diagnostic questions.
Surprisingly, a fluoroscopic image of a mouse (Swiss Nude) taken after applying a cyanine dye using a CCD camera showed a 1000 times greater fluorescent intensity as compared to a similarly-dosed ~porphyrin.
The use of the compounds of Formula I of other aspects of the present invention which constitutes aspects of the' present invention is particularly suitable for the visualization of tissue without pathological alterations, systemic diseases, tumours, blood vessels, atherosclerotic plaques, perfusion and diffusion.
The compounds of Formula I of other aspects of the present invention whose use constitutes aspects of the present invention are applied to the tissue in different ways.
Intravenous administration of tlhe dyes is particularly preferred.
Dosage may be quite different depending on the purpose of application. The goal to be achieved is a detectable concentration of dye in the tissue zone to be diagnosed, for which a concentration of 1 to 100 E.eg/ml in the tissue or in body fluids will mostly be sufficient. This concentration is reached by direct injection into small body cavities or small blood or lymph vessels, normally by applying 0.1 to 100 mg of the respective dye contained in 0.1 to 10 ml of vehicle liquid. In this case, 1 to 10 mg of dye are preferred.
Higher doses are mostly required to stain blood vessels or to detect specific tissues or structures after intravenous injection (greater than, or equal to, 100 mg).
The upper limit of dosage is only set by the tolerability of the respective substances and preparations.
Thus the invention in one' of its broad aspects is embodied in the use of compounds of the type B,-(F-W~)~" in which F represents a dye from the class of polymethine dyes, in particular, cyanine dyes. Merocyanine, styryl, oxonol and squarilium dyes may also be used. W is a structural element that contributes essentially to the hydrophilic of the whole molecule. Particularly preferre~J are compounds in which 1 represents the number 0, with their n-octanol/water distribution coefficient being smaller than 2 (n-octanol/0.01 M TRIS
buffer containing 0.9 % of sodium chloride, set to pH 7.4, both phases saturated against each other).
A biological detecting unit B may, for example, be an amino acid, a peptide, a CDR
(complementarity determining regions), an antigen, a hapten, an enzyme substrate, an enzyme cofactor, biotin, a carotenoid, a hormone, neurohormone, neurotransmitter, a growth factor, a lymphokin, a lectin, a toxin, a carbohydrate, an oligosaccharide, a polysaccharide, a dextran, an o~ligonucleotide made resistant to nucleases or a receptor-bonding pharmaceutical.
Compounds from the above-mentioned groups include, for example, ox:ytocins, vasopressins, angiotensins, melanocyte-stimulating hormones, somatostatins, thyrotrophin-releasing hormones, gonadotropin-releasing hormones, testosterones, estradiols, progesterones, cortisols, aldosterones, vitamin D, gastrins, secretins, somatropins, insulins, glucagons, calcitonin, STH-releasing hormones, prolacains, encephalins, dopamines, noradrenalines, serotonins, epinephrines, interleukins, angiogenins, thymopoietins, erythropoietins, fibrinogens, angiotensinogens, mecamylamines, ranitidine, cimetidine, lovastatines, isoproterenol derivatives or transferrin.
These substances facilitate accumulation in specific parts of the body by targeting the biological detecting unit throu~;h certain mechanisms. These mechanisms include bonding to extracellular structures, accumulation through various biological transport systems, recognition of cell surfaces or recognition of intracellular components.
Other compounds can be used to constitute aspects of the present invention in which B is a non-selectively bonding macromolecule, e.g., polylysine, polyethylene glycol, methoxypolyethylene glycol, p~ulyvinyl alcohol, dextran, carboxydextran or a cascade polymer-like structure that is covalently bonded to F.
The alkyl-, aryl- or aralk:yl residue with hydroxy groups contained in the compounds of the general Formula I of other aspects of the present invention whose use constitutes aspects of the present invention, are for example, 2-hydroxyethyl-, 2-hydroxypropyl-, 3-hydroxypropyl-, 4-hydroxyb~utyl-, 2,3-dihydroxypropyl-, 1,3-dihydroxyprop-2-yl-tris-(hydroxymethyl)-methyl-, 1,3,4-trihydroxybut-2-yl-glucosyl-, 4-(1,2-dihydroxyethyl)phenyl- or 2,4-, 2,5-, 3,5- or 3,4-dihydroxyphenyl residues.
An alkyl-, aryl- or aralkyl residue containing 1 to 3 carboxy groups may be, for example, a carboxymethyl-, carboxyethyl-, carboxypropyl-, carboxybutyl-, 1,2-dicarboxyethyl-, 1,3-dicarboxypropyl-, 3,5-dicarboyphenyl-, 3,4-dicarboxyphenyl-, 2,4-dicarboxyphenyl or 4-(1,2-dicarboxyethyl)-phenyl residue.
A sulphoalkyl residue preferably is a 2-sulphoethyl-, or a 3-sulphopropyl- or a 4-sulphobutyl residue.
Compounds in which W takes the position of R4 or Rg, R~ or R"' and R" or R'', and is also present in duplicate at positions R3/RS or R7/R'' are particularly preferred.
The dyes whose use constitutes aspects of the present invention absorb in the spectral range from 650 nm to 1200 nm~. The absorption coefficients of the compounds are ca.
100,000 1 mol-' cm' and more for one dye molecule. Fluorescent quantum efficiencies are greater than 5 % for all dyes used for fluorescent imaging.
Another important characteristic of the novel compounds of other aspects of the present invention is their hydrophilia marked by a n-octanol/water distribution coefficient smaller than 2.0 (distribution coefficient n-octanol/0.01 M TR1S buffer containing 0.9 %
sodium chloride, set to pH 7.4, both phases saturated against each other). The compounds do not have any distinct photosensitizing or phototoxic properties that would be undesirable in a diagnostic reagent. They are tolerated well, and discharged.
The hydrophilic behaviour of the novel compounds of other aspects of the present invention make them differ from dyes that have been previously proposed for use in in vivo diagnostics. Especially with cyanine dyes, fluorescent quantum efficiency valves, due to aggregation, drop dramatically in a hydrous environment, and are comparable:
to values measured in non-polar solvents; increased solubility in water and the space requirements of the hydrophilic groups suppress the formation of aggregates and micelles.
A group of preferred novel compounds of other aspects of the present invention shows little protein affinity; its pharmacokinetic behaviour is similar to that of insulin or saccharose, for example.
Surprisingly, these novel compounds of other aspects of the present invention showed diagnostically-sufficient accumulation in specific structures of the system, e.g;., in tumours, despite their simple molecular structure. When the dye has spread equally throughout the organism, its elimination, compared to the surrounding tissue, is delayed in tumour zones.
Tolerance of the substances is very good. Substances having LDS~, values greater than 0.5 mmol/kg body weight referred to a single dye molecule, are particularly preferred.
The novel compounds of other aspects of the present invention are characterized by great in vitro and in vivo stability, as well as photostability. When an aqueous solution thereof is allowed to stand in a daylit room, 98% of each of the novel compounds of other aspects of the present invention that are particularly preferred show no changes after 2 days, 70% show no changes after 12 days.
The photophysical and pharmacokinetic properties described of the novel compounds of other aspects of the present iinvention, also differ from those of the only cyanine dye approved for application in humans: indocyanine green (cardiogreen).
Preferred novel compounds of other aspects of the present invention are compounds of the general Formula I in which the 1-values of B are greater than, or equal to, 1, and preferably 1 or 2.
Cyanine dyes can be synthesized that have a great extinction coefficient when absorbing light at wavelengths from 650 to 1200 nm, and that fluoresce with great efficiency. Cyanine dyes of aspects of the present invention are mainly synthesized according to methods known from the literature, for example, F. M. Homer in The Cyanine Dies and Related Corn--pounds, John Wiley and Sons, New York, 1964;
Cytometry, 10 ( 1989) 3-10; 11 ( 1990) 418-430; 12 ( 1990) 723-30;
Bioconiu~ate Chem. 4 ( 1993) 105-11, Anal . Biochem_ 217 ( 1994) 197-204; Tetrahedron 45 ( 1989) 4845-66, European Patent Appl. 0 591 f>20 A 1.
The dye-biomolecule adducts of the compound of the general Formula 1: of other aspects of the present invention whose use constitutes aspects of the present invention are prepared by reacting a known compound F-W" prepared according to the procedures mentioned above with a biological detecting unit B.
The compound F-W" should therefore contain at least one, and preferably exactly one, grouping that can react covalently to an amine, hydroxy, aldehyde or sulphhydryl group on the biological detecting units. Such groupings are known from the literature and described in some detail, for example, in DE-OS 39 12 046.

Particularly preferred are isothiocyanate, isocyanate, and hydroxysuccinimide ester groupings or hydroxysulphosuc;cinimide ester groupings that are reactive to amino functions and form a thiourea, urea and amide bridge, as well as halogenacetyl and succinimide groupings that are reactive to sulphhydryl groups and form a thioether bridge.
Furthermore, carboxy groups with alcoholic functions may form ester linkages or ether structures using appropriate activating reagents (e.g., DCC), and aldehyde functions combined with hydrazines may result in imine structures.
The reactive groupings mentioned are added to the dyes of other aspects of the present invention of the general Formula I or their synthetic predecessors, or existing function groups are converted iinto the reactive groupings. The reactive groupings may be directly bonded to the dye syst~°m via so-called linker structures (e.g., alkyl clhains, aralkyl structures).
The F-W~ compounds arc' preferably reacted with the biological detecting units B in DMF or a hydrous environment or DMF/water mixtures at pH values between 7.4 and 10.
The molar proportion of dye and biomolecule (charging ratio) is determined using absorption spectrometry. Components that are not bound are separated by chromatography or filtering.
Macromolecules that have the appropriate functional groups may be coupled to the dyes in a similar way.
The substances may have quite different properties. Their molecular weight may be from a few hundreds to more than 100,000. The substances can be neutral or electrically charged. Salts of acid dyes andl physiologically acceptable bases, e.g., sodium, methyl glutamine, lysine, or salts containing lithium, calcium, magnesium, gadolinium in the form of cations.
The dye-biomolecule adducts thus gained excellently meet the above photophysical, toxicological, chemical and economic requirements.
These reagents are produced according to procedures known to persons skilled in the art, optionally by adding common adjuvants, diluents and the like. This includes physiologically tolerable electrolytes, buffers, detergents and substances for adjusting osmolarity and for improving stability and solubility, e.g., cyclodextrine.
Sterility of the preparations during their production and, in particular, before their application is to be ensured by taking the steps common in pharmaceutics.
(f~ DESCRIPTION OF THE FIGURES
In the accompanying drawings, FIG. 1 shows fluorescent light images (black-and-white) of a mouse (Swiss Nude) at various points in time after i.v. application of 3.8 mmol/kg body weight of 2-[7-[1,3-dihydro-3,3-dimethyl-5-(methoxycarbonyl)-1-(4-sulphobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxycarbonyl)-1-(4-sulphobutyl)-3H-indolium, sodium salt. In this FIG. 1, photographs A-E are right lateral images and photograph F is a posterior image, taken at the following times:
A: prior to application, B: 30 secs after application, C: 1 min after application, D: 10 mins after application, E: 1 h after application, and F: 18 h after application.
(g) AT LEAST ONE MODE FOR CARRYING OL1T THE INVENTION
The invention will now b~e explained by the following examples Example 1 Preparation of 5-[2-[(1,2-dicarboxyethyl)amino]-2-oxoethyl]-2-[7-[5-[2-[(1,2-dicarboxyethyl)amino]-2-oxoethyl]-1, 3-dihydro-3,3-dimethyl-1-(4-sulphobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulphobutyl)-3H-indolium, inner salt, potassium hydrogen salt.
Di-N-hydroxysuccinimide ester is prepared from 5-carboxymethyl-2-[7-[5-carboxy-methyl-1,3-dihydro-3,3-dimethyl-1-(4-sulphobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulphobutyl)- 3H-indolium, inner salt, potassium hydrogen salt according to known methods (C'.ytometry 11 ( 1990) 418-430) . 0.16 g ( 1.22 mmol) of aspartic acid in 1 ml of DMF are added to a solution of 0.5 g (0.51 mmol) of the disuccinimidyl ester in 5 ml DMF. The reaction mixture is stirred at room terrrperature for 48 h. The product is precipitated by adding ether, purified on RP-18 (LiChrop~repT"', 15-25 ~c, HZO:MeOH 99:1 to 1:1) and lyophilized. 0.27 g (51 %) of product are gained after drying for 24 hours at 500°C./0.01 mbar.
Analysis:
Calc.: C 54.43 H 5..'i4 N 5.40 O 24.68 S 6.18 K 3.77 Det.: C 54.04 H 5.81 N 5.22 S 6.13 K 3.85 Example 2 Preparation of 2-[7-[5-[2-[(I1-carboxy-2-oxo-1,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-1-andecyl)amino3-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-ethyl-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-I-(4-sulphobutyl)-3H-indolium, inner salt.
43 mg (0.65 mmol) of 85 % hydrazine hydrate in 1 ml of methanol are slowly added by dropping to a solution of 0.5 g (0.73 mmol) 2-[7-[5-(carboxymethyl)-1,3-dlihydro-3,3-dimethyl-1-ethyl-2H-indol-2-yliden]-I ,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulphobutyl)-3H-indolium-N-succinimidyl ester., inner salt in 5 ml of methanol (Cytometry 11 ( 1990) 418-430) at -10°C. and stirred for 2 hours at this temperature. The reaction mixture is evaporated under a vacuum to approx. 3 ml mixed with I ml of isopropanol and kept overnight at -20°C. The crystals that precipitate are sucked off and dried using the oil pump. The yield is 0.27 g (61 !~°) of tricarbocyanine carbonic acid hydrazide.
0.27 g (0.45 mmol) of the hydrazide are added under stirring to a solution of 0.21 g (0.51 mmol) of diethylene tria~mine pentaacetic monoethyl ester monoanhydride in 20 ml DMF and 0.2 ml triethylamine. The mixture is kept agitated at room temperature for 48 hours. The solvent is evaporat~°d at 0.2 mbar after filtering, the residue is mi:~ced up with CHZCIz, filtered off, and dried under high vacuum. The product gained is stirred up in 5 ml of 3M aqueous NaOH at room temperature for 4 hours. Then, a pH value of 2.0 is set using semiconcentrated HC1. 1 ml of isopropanol is added. After allowing the mixture to stand at 4°C. for 18 hours, crysvtals that have precipitated are sucked off and dried under high vacuum at 60°C. for 24 hours.
Yield: 0.23 g (52%) of granulate that glimmers dark red.
Analysis:
Calc.: C 59.32 H 6.60 N 9.88 O 20.96 S 3.23 Det.: C 54.15 H 6.70 N 9.50 S 3.19 Example 3 Preparation of 2-[7-[1,3-dlihydro-3,3-dimethyl-5-(2-[(methoxypolyoxyethylene)-amino]-2-oxoethyl]-1-(4-sulphobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)amino]-2-oxoethyl]-1-(4-sulphobutyl)-3H-indolium, sodium salt. A solution of 0.08 mmol of the N,N-disuccinimidyl ester from Example 1 in 1 ml of DMF is added to a solution of 800 mg of methoxypolyoxyethylene amine (ca. 0.16 mmol; average molar weight ca. 5000) in 10 ml of CHZCIZ and kept agitated at room temperature for 24 hours. The solid product that precipitates after adding ether is filtered off and purified by chromatography (SephadexTM G50 medium, H20 as eluent), yield approx. 58 % of l;reen-blue powder after lyophilization and drying above PZOS.
Average molar weight calc.: 10771, det. : 10820.
Example 4 Preparation of 2-[7-[1,3-dihydro-3,3-dimethyl-I-(4-sulphobutyl)-2H-indol-2 yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxypolyoxyethylene)aminocarbonyl-1 (4-sulphobutyl)-3H-indolium, sodium salt.
0.41 g (0.5 mmol) of 2-['7[1,3-dihydro-3,3-dirnethyl-1-(4-sulphobutyl-1-(4-sulphobutyl)-2H-indol-2-yl idea ]-1, 3 , 5-hepatrienyl]-3 , 3-dimethyl-5-carboxy-1 ~-(4-sulphobutyl)-3H-indolium-N-succinimidyl ester, sodium salt are stirred in an argon atmosphere together with 2.3 ~; of methoxy polyoxyethylene amine (0.46 mmol;
average molar weight: 5000) in 70 ml C"HZCIZ at room temperature for 18 hours. The solvent is reduced by half under vacuum and the product is isolated as described in Example 3. The yield is 2.1 g of product in the form of a green blue powder.
Average molar weight calc.: 5701, det.: 5795.
Example 5 Preparation of 3-(3-carboxypropyl)-2-[7-[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulphobutyl)-2H-indol-2-yl iden]-1, 3 , 5-heptatrienyl]-3-methyl-1-(4-sulphobutyl) - 3H-indolium, sodium salt.
6.5 g (50 mmol) of phenylhydrazine hydrochloride and 8.7 g (55 mmol',) of 5-methyl-6-oxoheptanoic acid are stirred in 50 ml of concentrated acetic acid at room temperature for 1 hour, and at 120°C. for 5 hours. After reducing by evaporation, the residue is mixed up with 20 ml of water, and the crystals that have precipitated are filtered off and dried using thc: oil pump.
This yields 9.6 g (83%) of brownish crystals that are suspended in 60 ml of dichlorobenzene and, after adding 11.6 g (85 mmol) of 1,4-butane sultone, heated for 8 hours to 150°C. After the mixture has cooled down to room temperature, 200 ml of acetone are added, and the precipitate is filtered off. It is suspended in ether, filtered off again after 18 hours of stirring, and dried using the oil pump. The yield is 10.7 g (70%) of 3-(3-carboxypropyl)-2,3-dime~thyl-1-(4-suiphobutyl)-3I-~-indolenin which is purified by chromatography (RP-18, LiChroprepTM, 15-25 ~, MeOH:H~O as eluent).
The indotricarbocyanine dye is prepared by heating 5.0 g ( 13.6 mmol) of indolenin and 1.9 g (6.8 mmol) of glutaconaldehyde dianilhydrochloride in 100 ml of .acetic anhydride for 30 minutes to 120°C., while adding 25 ml of conc. acetic acid and 2.3 g (27.6 mmol) of anhydrous sodium acetate. 500 ml of ether are added to the precipitate gained which is purified by chromatography (in portions of 1.0 g, RP-18, LiChroprepT"', 15-25 ,u, MeOH:HZO as eluent) and finally lyophilized. The yield is 2.5 g (45%) of the final product.

Analysis:
Calc.: C 60.13 H 6.28 N 3.42 O 19.54 S 7.83 Na 2.81 Det.: C 59.90 H 6.34 N 3.39 S 7.72 Na 2.78 Example 6 Preparation of 2-[[3-[[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulpho-butyl)2H-indol-2-yliden]met:hyl]2-hydroxy-4-oxo-2-cyclobuten-1-yliden]mEahyl]-1,1-dimethyl-3-ethyl-1H-benz(e;lindolium, inner salt, 3.65 g (10.0 mmol) of 3-ethyl-1,1,2-trimethyl-1H-l:~enz(e)indoliumiodide are added to a solution of 1.36 g (8.0 mmol) squaric diethyl ester and 1.6 ml triethylamine in 12 ml of ethanol that is heated up to 70°C. After 10 minutes of stirring at 80°C., the mixture is cooled down to 0°C. The precipitated, red-coloured crystals are filtered off, washed with ether, and dried under vacuum. Purification by chromatography on silica gel (CHZCIZ:AcOH 9:1 to 7:3) yields 1.33 g (46%) of 2-ethoxy-1-[(3-ethyl-1,1-dimethyl-1H-benz(e)indol-2-ylid~en)-methyl]-cyclobuten-3,4-dion.
This substance is suspended in 15 ml of boiling ethanol and mixed under stirring with 0.5 ml of 40% NaOH. The solution gained is stirred for 5 minutes at 80°C'. and mixed with 5 ml of 2N HCl after cooling down to room temperature. The 1-[(3-ethyl-1,1-dimethyl-1H-benz(e)indol-2-yliden)-methyl]-2-hydroxycyclobutene-3,4-dion (1.30 g) that precipitates after evaporating is filtered, dried and used for the next step of synthesis without being purified.
The squarain dye is prepared by reacting 1.30 g (3.9 mmol) of the squaric acid derivative gained with 1.43 g (3.9 mmol) of 3-(3-carboxypropyl)-2,3-dime~thyl-1-(4-sulpho-butyl)-3H-indolenin. The components obtained are heated for 18 hours in 80 ml of toluene and 80 ml 1-butanol at the water separator and then treed from solvents under vacuum. The residue is mixed with ether, and the crystals that have formed are filtered off after 16 hours of stirring at room temperature, and purified by chromatography (RP-18, LiChroprepT"~', 15-25 ~, MeOH:H20 as eluent), yield: 0.95 g (36%).

Analysis:
Calc.: C 68.60 H 6.20 N 4.10 O 16.40 S 4.70 Det.: C 68.25 H 6.35 N 4.04 S 4.59 Example 7 Preparation of 2-[7-[1,3-dihydro-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulphobutyl)-2H-indol-2-yliden]-I ,3,5-heptatrienyl]-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulphobutyl)-3H-indolium, sodium salt.
2.0 g (6.4 mmol) of 2,3,3-trimethyl-3H-indol-5-yl acetic succinimid;yl ester in 50 ml CH,Cl2 are mixed with 0.84 g (6.4 mmol) of 4-aminomethyl-2,2-dimethyl-1,3-dioxolane.
After 5 hours of stirring at room temperature, the mixture is poured on 100 ml of water and extracted with CHzC(Z; the organic phases are evaporated. After chromatographic purification (silica gel CHzCh:MeOH 98:2) 1.86 g (88%) of the amide are gained which are stirred for 12 hours in :?0 ml of dichlorobenzene and 1.36 g ( 10.0 mmol) of 1,4-butane sultone at room temperature, and at 100°C. The granulate that is formed after stirring the mixture up with 50 ml of acetone is filtered off and purified by chromato~;raphy (RP-18, LiChroprepT"', 15-25 ,u, MeOH:HzO as eluent). The yield is 0.85 g (28%
referred to the parent compound) of 5-[2-~(2,2-dimethyl-1,3-dioxa-4-cyclopentyl)methyl]amino-2-oxoethyl]-2, 3 , 3-trimethyl-1. -(4-sulphobutyl)-3 H-indolenin.
The reaction to produce the dye is similar to Example 4. The substance is heated for minutes to 120°C. The crude product is stirred at room temperature in 5 ml MeOH by adding 100 mg toluene-p-sulphonic acid for 16 hours; insoluble parts are separated. The filtrate is then kept at -20°(~'. after adding 3 ml of isopropanol. The powder that precipitates is purified by chromatography (RP-18, LiChroprepT"'', 15-25 ~, MeOH:Hf20 as eluent), lyophilized and dried for 24 hours at 50°C./0.01 mbar.
Yield: 0.32 g (37%).

Analysis:
Calc.: C 56.70 H 6.45 N 5.88 O 20.14 S 6.73 K 4.10 Det.: C 56.39 H 6.88 N 5.67 S 6.58 K 3.93 Example 8 2-[7-[ 1, 3-dihydro-3 , 3-dinnethy 1-5-(methoxycarbony 1)-1-(4-sulphobuty 1)-:2,H-indol-2-yliden]-1,3,5-heptatrienyl)-3.,3-dimethyl-5-(methoxycarbonyl)-1-(4-sulphobutyl)-3H-indolium, sodium salt at a dose of 3.8 ~c mol/kg body weight was applied intravenously to an anaesthetised, tumour-bearing mouse (Swiss Nude, tumour LS 174 T at the:
right hindleg).
The laser-induced fluorescent images were taken prior to, and at various points in time after, applying the substance with a l7uorescence imager (at Physikalisch-Technische Bundesanstalt, Berlin Charlottenburg). Radiation was excited using monochromatic laser light at 740 nm by decoupling the radiation via a fibre optical waveguide system. Exciting radiation below 740 nm was removed using a cutoff filter. The laser-induced fluorescence light above 740 nm was recorc'led using a CCD camera (Charge Coupled Device), and data was stored in the form of black-and-white images.
The sequence of shots shown in FIG. 1 clearly shows a general increase of fluorescent intensity after the substance was applied (A, B). A uniform distribution of intensity can be observed after 30 seconds, with values being increased in the hepatic and pulmonary regions and in the tumour (B). With more time elapsing (up to 1 h) (C,D,E), the substance spreads more and more throughout the animal. After 18 hours, a clearly increased fluorescent intensity can be observed in the tumour (right hindleg) as compared with the rest of the body.
Figure 1 shows fluorescent light images (black-and-white) of a mouse (Swiss Nude) at various points in time after i.v. application of 3.8 mmol/kg body weight o:f 2-[7-[1,3-37 a dihydro-3,3-dimethyl-5-(methoxycarbonyl-1-(4-sulphobutyl)-ZH-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5~-(methoxycarbonyl)-1-(4-sulphobutyl)-3H-indol.ium, sodium salt A-E: right lateral images, F: posterior image A: prior to application, B: 30 secs, C: 1 min, D: 10 mins, E: 1 h after application, F: 18 h after application.

Claims (24)

1. An in-vivo diagnostic near infrared radiation method comprising:

(a) administering a compound of the general formula I

B 1 --(F-Wn)m, (I) wherein I represents a number from 0 to 6, n represents a number from 0 to 10 and m represents a number from 1 to 100;

B is a biological detecting unit having a molecular weight of up to 30,000 that bonds to specific cell populations or selectively to receptors, or accumulates in tissues or tumours, or generally stays in the blood, or is a macromolecule that bonds non-selectively;

F represents a dye showing maximum absorption in the range of 650 to 1200 nm having the general formula IIb wherein R3 to R12 are same or different, each independently representing a hydrogen atom, a residue B or W, or an alkyl or alkenyl residue containing up to 6 carbon atoms or an aryl or aralkyl residue containing up to 9 carbon atoms, said alkyl, alkenyl, aryl or aralkyl residue optionally carrying an additional residue W or W to each pair of adjacent residues R3 to R10 are annealed, with due regard for the interspersed atoms, 5- to 6-member rings that may be saturated, unsaturated or aromatic, and that may optionally carry residue R, R is a halogen atom, a hydroxy, carboxy, acetoxy, amino, nitro, cyano or sulfonic acid group or an alkyl, alkenyl, hydroxyalkyl, carboxyalkyl, alkoxy, alkoxycarbonyl, sulfoalkyl, alkylamino, dialkylamino or halogenalkyl residue containing up to 6 carbon atoms, an aryl, alkylaryl hydroxyaryl, carboxyaryl, sulfoaryl, arylamino, diarylamino, nitroaryl or halogenaryl residue containing up to 9 carbon atoms, s and t independently represent the numbers 0 to 1 on the condition that s and t do not represent the number 1 at the same time, X and Y are same or different, each independently representing an O, S, Se or Te or a -C(CH3)2 -, -CH=CH- or -CR13 R14 - fragment, wherein R13 and R14 independently represent a hydrogen atom, a residue B or W, or an alkyl or alkenyl residue containing up to 6 carbon atoms or an aryl or aralkyl residue containing up to 9 carbon atoms, the alkyl, alkenyl, aryl or aralkyl residue optionally having an additional residue W;

W represents a hydrophilic group that improves water-solubility, with the n-octanol-water distribution coefficient of the compound according to formula I
being less than or equal to 2.0 for 1=0;

or its physiologically tolerable salts;

(b) irradiating the compound with light in the near infrared range; and (c) recording the radiation produced by the compound.

2. A method according to claim 1, wherein B within the general formula I is selected from the group consisting of an amino acid, a peptide, a complementarity determining region, an antigen, a hapten, an enzyme substrate, an enzyme cofactor, biotin, a carotinoid, a hormone, a neurohormone, a neurotransmitter, a growth factor, a lymphokin, a lectin, a toxin, a carbohydrate, an oligosaccharide, a polysaccharide, a dextran, an oligonucleotide and a receptor-bonding pharmaceutical.

3. A method according to claim 1 or 2, wherein W within the general formula I
is selected from the group consisting of (i) a carboxy or sulfonic acid group;
(ii) a carboxyalkyl group; (iii) an alkoxycarbonyl or alkoxyoxoalkyl group containing up to 12 carbon atoms; (iv) a residue of the general formula III

-(CH2)a -O-Z or (-CH2 -CH2 -O)a -Z (III) wherein a represents the numbers 0 to 6;

Z represents a hydrogen atom or an alkyl residue containing 3 to 6 C atoms that includes 2 to n-1 hydroxy groups, with n being the number of C atoms, or an aryl or aralkyl residue containing 6 to 10 C atoms and having 2 to 4 additional hydroxy groups, or an alkyl residue containing 1 to 6 C atoms and having 1 to 3 additional carboxy groups, or an aryl residue containing 6 to 9 C atoms and having 1 to 3 additional carboxyl groups, or an aralkyl residue or a nitroaryl or a nitroaralkyl residue containing 6 to 15 C atoms, or a sulfoalkyl residue containing 2 to 4 C atoms having 1 to 3 additional carboxy groups, or represents a residue of the general formula IIIa or IIIb or a residue of the general formula IIIc -(CH2)o -(CO)p -NRl -(CH2)S -(NH-CO)q -R2 (IIIC) wherein o and s independently represent the numbers, 0, 1, 2, 3, 4, 5 or 6, p and q independently represent 0 to 1, R1 and R2 independently represent a residue Z except the substituents of the general formulas IIIa and IIIb, or independently represent a residue of the general formulas IIId or IIIe;

on the condition that p and q=1; and (v) a residue of the general formula IIIc.

4. A cyanine dye of the general formula V

where Q is a fragment selected from the group consisting of where R30 represents a hydrogen atom, a hydroxy group, a carboxy group, an alkoxy residue containing 1 to 4 carbon atoms or a chlorine atom, b is an integer 2 or 3, R31 represents a hydrogen atom or an alkyl residue containing 1 to 4 carbon atoms, X and Y independently represent an -O-, -S-, -CH-CH- or -C(CH2 R32)(CH2 R33)- fragment each, R20 to R29, R32 and R33 independently represent a hydrogen atom, a hydroxy group, a carboxy-, a sulfonic acid residue or a carboxyalkyl-, alkoxycarbonyl or alkoxyoxoalkyl residue containing up to 10 C atoms or a sulfoalkyl residue containing up to 4 C atoms, or a non-selectively bonding macromolecule or a residue of the general formula VI

-(O)v -(CH2)o -CO-NR34 -(CH2)s -NH-CO)q -R35 (VI) on the condition that, where X and Y are O, S, -CH=CH- or -C(CH3)2 - at least one of the residues R20 to R29 corresponds to a non-selectively bonding macromolecule or a residue of the general formula VI, where o and s equal 0 or independently represent an integer between 1 and 6, q and v independently represent 0 or 1, R34 represents a hydrogen atom or a methyl residue, R35 represents an alkyl residue containing 3 to 6 C atoms and comprising 2 to n-1 hydroxy groups, with n being the number of C atoms, or an alkyl residue containing 1 to 6 C atoms that carries 1 to 3 additional carboxy groups, an aryl residue containing 6 to 9 C
atoms or arylalkyl residue containing 7 to 15 C atoms, or a residue of the general formula IIId or IIIe on the condition that q is 1, or a non-selectively bonding macromolecule, or R20 and R21, R21 and R22, R22 and R23, R24 and R25, R25 and R26, R26 and R27, together with the interspersed carbon atoms, form a 5- or 6-member aromatic or saturated annelled ring, and its physiologically tolerable salts.

5. An agent for an in-vivo diagnositic method based on near infrared radiation comprising the cyanine dye according to claim 4 together with physiologically acceptable adjuvants, substrates, and/or diluents.

6. A cyanine dye according to claim 4 or 5, wherein said dye is selected from the group consisting of 5-[2-[(1,2-dicarboxyethyl)amino]-2-oxoethyl]-2-[7-[5-[2-[(1,2-dicarboxyethyl)amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, inner salt, potassium hydrogen salt;
2-[7-[5-[2-[(11-carboxy-2-oxo-1,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-1-undecyl)amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-ethyl-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, inner salt;
2-[7-[1,3-dihydro-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)-amino]-2-oxoethyl]-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)amino]-2-oxoethyl]-1-(4-sulfobutyl)-3H-indolium, sodium salt;
2-[7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxypolyoxy-ethylene)aminocarbonyl-1-(4-sulfobutyl)-3H-indolium, sodium salt;
3-(3-carboxypropyl)-2-[7-[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3-methyl-1-(4-sulfobutyl)-indolium, sodium salt;
2-[[3-[[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulfobutyl)2H-indol-2-yliden]methyl]-2-hydroxy-4-oxo-2-cyclobuten-1-yliden]methyl]-1,1-dimethyl-3-ethyl-1H-2-benz(e)indolium, inner salt; and 2-[7-[1,3-dihydro-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, sodium salt.

7. A method of diagnosing diseased tissue comprising administering the cyanine dye claimed in any one of claims 4 to 6, irradiating the diseased tissue with light in the visible to near infrared range and recording fluorescent radiation produced from the cyanine dye.

8. An in-vivo diagnostic near infrared radiation method comprising:
(a) administering a compound of the general formula I
B1 ~(F~W n)m (I) wherein 1 represents a number from 0 to 6, n represents a number from 0 to 10 and m represents a number from 1 to 100;
B is a biological detecting unit having a molecular weight of up to 30,000 that bonds to specific cell populations or selectively to receptors, or accumulates in tissues or tumours, or generally stays in the blood, or is a macromolecule that bonds non-selectively;
F represents a dye showing maximum absorption in the range of 650 to 1200 nm;
W represents a hydrophilic group that improves water-solubility, with n-octanol-water distribution coefficient of the compound according to formula I being less than or equal to 2.0 for 1=0;
or its physiologically tolerable salts;
(b) irradiating the compound with light in the near infrared range; and (c) recording the radiation produced by the compound, wherein F represents a cyanine dye of the general formula IIa wherein r represents the numbers 0, 1 or 2, wherein, for r=2, the respective L6 and L7 groups that occur in duplicate is the same or different, L1 to L7 are the same or different, each independently representing a CH or CR
group, where R is a halogen atom, a hydroxy, carboxy, acetoxy, amino, nitro, cyano, or sulfonic acid group or an alkyl, alkenyl, hydroxyalkyl, carboxyalkyl, alkoxy, alkoxycarbonyl, sulfoalkyl, alkylamino, dialkylamino or halogenalkyl group containing up to 6 carbon atoms, aryl, alkylaryl hydroxyaryl, carboxyaryl, sulfoaryl, arylamino, diarylamino, nitroaryl or halogenaryl group containing up to 9 carbon atoms, or where R
represents a bond that bonds to another group R and forms a 4- to 6-member ring together with the interspersed groups L1 to L7, or two of L1 to L7 that are linked via a --CO-- group, R3 to R12 are the same or different, each independently representing a hydrogen atom, a group B or W, or an alkyl or alkenyl group containing up to 6 carbon atoms or an aryl or aralkyl group containing up to 9 carbon atoms, said alkyl, alkenyl, aryl or aralkyl group, optionally carrying an additional group W, or to each pair of adjacent groups R3 to R10 are annealed with due regard for the interspersed C atoms, 5-to 6-member rings that are saturated, unsatured or aromatic, and that optionally carry an additional group R, and X and Y are the same or different, each independently representing an O, S, Se or Te or a ~C(CH3)2~, ~CH=CH~ or ~CR13R14~ group, where R13 and R14 independently represent a hydrogen atom, a group B or W, or an alkyl or alkenyl group containing up to 6 carbon atoms or an aryl or aralkyl group containing up to 9 carbon atoms, the alkyl, alkenyl, aryl or aralkyl group optionally carrying an additional group W.

9. A method according to claim 8, wherein B within the general formula I is selected from the group consisting of an amino acid, a peptide, CDR (complementarity determining region), an antigen, a hapten, an enzyme substrate, an enzyme cofactor, biotin, a carotinoid, a hormone, a neurohormone, a neurotransmitter, a growth factor, a lymphokin, a lectin, a toxin, a carbohydrate, an oligosaccharide, a polysaccharide, a dextran, an oligonucleotide and a receptor-bonding pharmaceutical.

10. A method according to claim 8 or 9, wherein W within the general formula I
is selected from the group consisting of (i) a carboxy or sulfonic acid group;
(ii) a carboxyalkyl group; (iii) an alkoxycarbonyl or alkoxyoxoalkyl group containing up to 12 carbon atoms, (iv) a group of the general formula III
~(CH2)a ~O~Z or (~CH2~CH2~O)a~Z (III) wherein a represents the numbers 0 to 6, Z represents a hydrogen atom or an alkyl group containing 3 to 6 C atoms having 2 to n-1 hydroxy groups, with n being the number of C atoms or an aryl or aralkyl group containing 6 to 10 C atoms and having 2 to 4 additional hydroxy groups, or an alkyl group containing 1 to 6 C atoms and having 1 to 3 additional carboxy groups, or an aryl group containing 6 to 9 C atoms and having 1 to 3 additional carboxyl groups or an aralkyl group or a nitroaryl or a nitroaralkyl group containing 6 to 15 C atoms or a sulfoalkyl group containing 2 to 4 C atoms having 1 to 3 additional carboxy groups; a group of the general formulas III a or III b a group of the general formula III c ~(CH2)o~(CO)p~NR1~(CH2)s~(NH-CO)q~R2 (III c) wherein o and s independently represent the numbers 0, 1, 2, 3, 4, 5 or 6, p and q independently represent 0 or 1, R1 and R2 independently represent a group Z except the substituents of the general formulas III a and III b, or independently represent a group of the general formulas III
d or III e wherein p and q=1; and (v) a group of the general formula III.

11. A dye having the formula I:
B L-(F-W n)m (I) wherein L represents a number from 1 to 6;
n represents a number from 0 to 10;
m represents a number from 1 to 100;
B is a biological detecting unit having a molecular weight of up to 30,000;
W is a hydrophilic group that improves water solubility;
F is a dye showing maximum absorption in the range of 650 to 1200 nm having the general formula V:
wherein Q represents a fragment selected from the group consisting of:

wherein R30 is selected from a hydrogen atom, a hydroxy group, a carboxy group, an alkoxy residue containing 1 to 4 carbon atoms, and a chlorine atom;
b is 2 or 3;
R31 is selected from a hydrogen atom and an alkyl residue containing 1 to 4 carbon atoms;
X and Y are independently selected from -O-, -S-, -CH=CH-, and -C(CH2R32)(CH2R33)-;
R20 to R29, R32, and R33 are each independently selected from the group consisting of a hydrogen atom, a hydroxy goup, a carboxylic acid, a sulfonic acid residue, a carboxyalkyl residue containing up to 10 carbon atoms, an alkoxycarbonyl residue containing up to 10 carbon atoms, an alkoxyoxoalkyl residue containing up to 10 carbon atoms, a sulfoalkyl residue containing up to 4 carbon atoms, a 5- or 6-member aromatic or saturated annellled ring comprising R20 and R21 and interspersed carbon atoms, a 5- or 6-member aromatic or saturated annelled ring comprising R21 and R22 and interspersed carbon atoms, a 5- or 6-member aromatic or saturated annelled ring comprising R22 and R23 and interspersed carbon atoms, a 5- or 6-member aromatic or saturated annelled ring comprising R24 and R25 and interspersed carbon atoms, a 5- or 6-member aromatic or saturated annelled ring comprising R25 and R26 and interspersed carbon atoms, and a 5- or 6-member aromatic or saturated annelled ring comprising R26 and R27;
on the condition that, where X and Y are selected from -O-, -S-, -CH=CH-, and -C(CH3)2-, at least one of the residues R20 to R29 is a non-selectively bonding macromolecule or residue of formula VI
~(O)v~(CH2)o~CO~NR34~(CH2)s~(NH~CO)q~R35 (VI);
where o and s equal 0 or independently represent an integer between 1 and 6, q and v independently represent 0 or 1, R34 represents a hydrogen atom or a methyl residue, R35 represents an alkyl residue containing 3 to 6 C atoms and comprising 2 to n-1 hydroxy groups, with n being the number of C atoms, or an alkyl residue containing 1 to 6 C atoms that carries 1 to 3 additional carboxy groups, an aryl residue containing 6 to 9 C atoms or arylalkyl residue containing 7 to 15 C atoms, or a residue of the general formula IIId or IIIe on the condition that q is 1; and;

51a pharmaceutically acceptable salts thereof.

12. A dye having the formula I:
B L-(F-W n)m (I) wherein L represents a number from 1 to 6;
n represents a number from 0 to 10;
m represents a number from 1 to 100;
B is a biological detecting unit having a molecular weight of up to 30,000;
W is a hydrophilic group that improves water solubility;
F represents a dye showing maximum absorption in the range 650 to 1200 nm having the general formula V:
wherein Q represents a fragment selected from the group consisting of wherein R30 is selected from a hydrogen atom, a hydroxy group, a carboxy group, an alkoxy residue containing 1 to 4 carbon atoms, and a chlorine atom;
b is 2 or 3;
R31 is selected from a hydrogen atom and an alkyl residue containing 1 to 4 carbon atoms;
X and Y are independently selected from -O-, -S-, -CH=CH-, and -C(CH2R32)(CH2R33)-;
R20 to R29, R32, and R33 are each independently selected from the group consisting of a hydrogen atom, a hydroxy group, a carboxylic acid residue containing up to carbon atoms, a sulfonic acid residue containing up to 10 carbon atoms, a carboxyalkyl residue containing up to 10 carbon atoms, an alkoxycarbonyl residue containing up to 10 carbon atoms, an alkoxyoxoalkyl residue containing up to 10 carbon atoms, a sulfoalkyl residue containing up to 4 carbon atoms, a 5- or 6-member aromatic or saturated annelled ring comprising R20 and R21 and interspersed carbon atoms, a 5- or 6-member aromatic or saturated annelled ring comprising R21 and R22 and interspersed carbon atoms, a 5- or 6-member aromatic or saturated annelled ring comprising R22 and R23 and interspersed carbon atoms, a 5- or 6-member aromatic or saturated annelled ring comprising R24 and R25 and interspersed carbon atoms, a 5- or 6-member aromatic or saturated annelled ring comprising R25 and R26 and interspersed carbon atoms, a 5- or 6-member aromatic or saturated annelled ring comprising R26 and R27 and a non-selectively bonding macromolecule or residue of formula VI:
-(O)v-(CH2)o-CO-NR34-(CH2)s-(NH-CO)q-R35 (IV) on the condition that, where X and Y are selected from -O-, -S-, -CH=CH-, and -C(CH3)2-, at least one of the residues R20 to R29 is a non-selectively bonding macromolecule or residue of formula VI;
wherein o and s are independently an integer between 0 and 6;
q and v are independently 0 or 1;
R34 is selected from a hydrogen atom and a methyl residue, and R35 is selected from the group consisting of an alkyl residue containing 3 to carbon atoms and comprising from 2 to z-1 hydroxy groups with z being the number of carbon atoms, an alkyl residue containing 1 to 6 carbon atoms comprising from 1 to 3 carboxy groups, an aryl residue containing 6 to 9 carbon atoms, an arylalkyl residue containing 7 to 15 carbon atoms, a residue of formula III d, and a residue of formula III e, wherein when R35 is formula III d or III e, q is 1; and pharmaceutically acceptable salts thereof.

13. The dye of claim 2, wherein the dye compasses one or more of the cyanine dyes selected from the group consisting of 5-[2-[(1,2-dicarboxyethyl)amino]-2-oxoethyl]-2-[7-[5-[2-(1,2-dicarboxyethyl )amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl)-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, inner salt, and potassium hydrogen salt;
2-[7-[5-[2-[(11-carboxy-2-oxo-1,4,7,10-tetraaza-4,7,10-tri(carboxymethyl)-1 -andecyl)amino]-2-oxoethyl]-1,3-dihydro-3,3-dimethyl-1-ethyl-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, and inner salt;
2-[7-[1,3-dihydro-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)-amino]-2-oxoethyl]-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-[2-[(methoxypolyoxyethylene)amino]-2-oxoethyl]-1-(4-sulfobutyl)-3H-indolium, sodium salt;
2-[7-[1,3-dihydro-3,3-dimethyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-3,3-dimethyl-5-(methoxypolyoxyethylene)aminocarbonyl-1-(4-sulfobutyl)-3H-indolium, and sodium salt;

3-(3-carboxypropyl)-2-[7-[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl)-3-methyl-1-(4-sulfobutyl)-3 H-indolium, and sodium salt;
2-[[3-[[3-(3-carboxypropyl)-1,3-dihydro-3-methyl-1-(4-sulfobutyl)2H-indol-2 -yliden]methyl]2-hydroxy-4-oxo-2-cyclobuten-1-yliden]methyl]-1,1-dimethyl-3-ethyl-1H-benz(e)indolium, and inner salt; and 2-[7-[1,3-dihydro-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl)-3,3-dimethy 1-1-(4-sulfobutyl)-2H-indol-2-yliden]-1,3,5-heptatrienyl]-5-[2-[(2,3-dihydroxypropyl)amino]-2-oxoethyl]-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium, and sodium salt.

14. The dye of claim 11, wherein the dye has an n-octanol/water distribution coefficient of less than 2Ø

15. The dye of claim 12, wherein the dye has an n-octanol/water distribution coefficient of less than 2Ø

16. The dye of claim 11, wherein the dye has an LD50 value of greater than 0.5 mmol/kg body weight.

17. The dye of claim 12, wherein the dye has an LD50 value of greater than 0.5 mmol/kg body weight.

18. A method of diagnosing diseased tissue comprising administering the dye claimed in any one of claims 11, 14 and 16, irradiating the diseased tissue with light in the visible to near infrared range, and recording fluorescent radiation produced from the dye.

19. A method of diagnosing diseased tissue comprising administering the dye claimed in any one of claims 12, 13, 15 and 17, irradiating the diseased tissue with light in the visible to near infrared range, and recording fluorescent radiation produced from the dye.

20. An agent for an in vivo diagnostic method based on near infrared radiation comprising the dye of any one of claims 11, 14 and 16, and one or more selected from physiological acceptable adjuvants, substitutes and diluents.

21. An agent for an in vivo diagnostic method based on near infrared radiation comprising the dye of any one of claims 12, 13, 15 and 17, and one or more selected from physiological acceptable adjuvants, substitutes and diluents.

22. A cyanine dye as represented by the general formula IIa:
wherein r represents the numbers 0, 1 or 2, wherein, for r=2, the respective L6 and L7 groups that occur in duplicate may be the same or different, L1 to L7 are the same or different, each independently representing a CH or CR group, where R is a halogen atom, a hydroxy, carboxy acetoxy, amino, nitro, cyano or sulfonic acid group or an alkyl, alkenyl, hydroxyalkyl, carboxyalkyl, alkoxy, alkoxycarbonyl, sulfoalkyl, alkylamino, dialkylamino or halogenalkyl group containing up to 6 carbon atoms, aryl, alkylaryl, hydroxyaryl, carboxyaryl, sulfoaryl, arylamino, diarylamino, nitroaryl or halogenaryl group containing up to 9 carbon atoms, or where R represents a bond that bonds to another group R and forms a 4- to 6-member ring together with the interspersed groups L1 to L7, or two of L1 to L7 that are linked via a -CO- group, R3 to R12 are the same or different, each independently representing a hydrogen atom, a group B or W, or an alkyl or alkenyl group containing up to 6 carbon atoms or an aryl or aralkyl group optionally carying an additional group W, or to each pair of adjacent groups R3 to R10 are annealed, with due regard for the interspersed C atoms, 5- to 6-member rings that may be saturated, unsaturated or aromatic, and that optionally carry an additional group R, and X and Y are the same or different, each independently representing an O, S, Se or Te or a -C(CH)2-, -CH=CH- or -CR13R14- group, where R13 and R14 independently represent a hydrogen atom, a group B or W, or an alkyl or alkylene group containing up to 6 carbon atoms or an aryl or aralkyl group containing up to 9 carbon atoms, the alkyl, alkenyl, aryl or aralkyl group optionally carrying an additional group W, wherein B is a biological detecting unit having a molecular weight of up to 30,000 that bonds to specific cell populations or selectively to receptors, or accumulates in tissues or tumors, or generally stays in the blood, or is a macromolecule that bonds non-selectively and W is a hydrophilic group that improves water-solubility selected from the group consisting of (i) a carboxylic acid group (ii) a carboxyalkyl group (iii) an alkoxycarbonyl or alkoxyoxoalkyl group containing up to 12 carbon atoms, (iv) a group of the general formula III
-(CH2)n-O-Z or -(CH2-CH2-O)~-Z (III) wherein a represents the numbers 0 to 6, Z represents a hydrogen atom or an alkyl group containing 3 to 6 C atoms having 2 to n-1 hydroxy groups, with n being the number of C atoms or an aryl or aralkyl group containing 6 to 10 C atoms and having 2 to 4 additional hydroxy groups, or an alkyl group containing 1 to 6 C atoms and having 1 to 3 additional carboxy groups, or an aryl group containing 6 to 9 C atoms and having 1 to 3 additional carboxyl groups or an aralkyl group or a nitroaryl or a nitcoaralkyl group containing 6 to 15 C atoms;
a group of the general formulas IIIa or IIIb a group of the general formula IIIc -(CH2)o-(CO)p-NR1-(CH2)s-(NH-CO)q-R2 (IIIc) wherein o and s independently represent the numbers 0, 1, 2, 3, 4, 5 or 6, p and q independently represent 0 or 1, R1 and NR2 independently represent a group Z except the substituents of the general formulas IIIa and IIIb, or independently represent a group of the general formulas IIId or IIIe wherein p and q = 1; and (v) a group of the general formula III as defined above.

23. A near infrared imaging agent comprising a cyanine dye represented by the general formula IIa:
wherein r represents the numbers 0, 1 or 2, wherein, for r=2, the respective L6 and L7 groups that occur in duplicate may be the same or different, L1 to L7 are the same or different, each independently representing a CH or CR group, where R is a halogen atom, a hydroxy, carboxy acetoxy, amino, nitro, cyano or sulfonic acid group or an alkyl, alkenyl, hydroxyalkyl, carboxyalkyl, alkoxy, alkoxycarbonyl, sulfoalkyl, alkylamino, dialkylamino or halogenalkyl group containing up to 6 carbon atoms, aryl, alkylaryl, hydroxyaryl, carboxyaryl, sulfoaryl, arylamino, diarylamino, nitroaryl or halogenaryl group containing up to 9 carbon atoms, or where R represents a bond that bonds to another group R and forms a 4- to 6-member ring together with the interspersed groups L1 to L7, or two of L1 to L7 that are linked via a -CO- group, R3 and R2 are the same or different, each independently representing a hydrogen atom, a group B or W, or an alkyl or alkenyl group containing up to 6 carbon atoms or an aryl or aralkyl group optionally carrying an additional group W, or to each pair of adjacent groups R3 and R10 are annealed, with due regard for the interspersed C
atoms, 5- to 6- member rings that may be saturated, unsaturated or aromatic, and that optionally carry an additional group R, and X and Y are the same or different, each independently representing an O, S, Se or Te or a -C(CH3)2-, -CH=CH- or -group, where R13 and R14 independently represent a hydrogen atom, a group B or W, or an alkyl or alkylene group containing up to 6 carbon atoms or an aryl or aralkyl group containing up to 9 carbon atoms, the alkyl, alkenyl, aryl or aralkyl group optionally carrying an additional group W, on the condition that, where X and Y are selected from -O-, -S-, -CH=CH-, and -C(CH3)2-, at least one of the residues R3 to R12 is a non-selectively bonding macromolecule or residue of formula VI
~(O)v~(CH2)o~CO~NR34~(CH2)s~(NH-CO)q~R35 (VI);
where o and s equal 0 or independently represent an integer between 1 and 6, q and v independently represent 0 or 1, R34 represents a hydrogen atom or a methyl residue, R35 represents an alkyl residue containing 3 to 6 C atoms and comprising 2 to n-1 hydroxy groups, with n being the number of C atoms, or an alkyl residue containing 1 to 6 C atoms that carries 1 to 3 additional carboxy groups, an aryl residue containing 6 to 9 C atoms or arylalkyl residue containing 7 to 15 C atoms, or a residue of the general formula IIId or IIIe 60a on the condition that q is 1, wherein B is a biological detecting unit having a molecular weight of up to 30,000 that bonds to specific cell populations or selectively to receptors, or accumulates in tissues or tumors, or generally stays in the blood, or is a macromolecule that bonds non-selectively and W is a hydrophilic group that improves water-solubility;
wherein the cyanine dye is coupled to a hydrophilic group.

24. A near infrared imaging agent comprising a cyanine dye represented by the general formula IIa:
wherein r represents the numbers 0, 1 or 2, wherein, for r=2, the respective L6 and L7 groups that occur in duplicate may be the same or different, L1 to L7 are the same or different, each independently representing a CH or CR group, where R is a halogen atom, a hydroxy, carboxy acetoxy, amino, nitro, cyano or sulfonic acid group or an alkyl, alkenyl, hydroxyalkyl, carboxyalkyl, alkoxy, alkoxycarbonyl, sulfoalkyl, alkylamino, dialkylamino or halogenalkyl group containing up to 6 carbon atoms, aryl, alkylaryl, hydroxyaryl, carboxyaryl, sulfoaryl, arylamino, diarylamino, nitroaryl or halogenaryl group containing up to 9 carbon atoms, or where R represents a bond that bonds to another group R and forms a 4- to 6-member ring together with the interspersed groups L1 to L7, or two of L1 to L7 that are linked via a -CO- group, R3 and R12 are the same or different, each independently representing a hydrogen atom, a group B or W, or an alkyl or alkenyl group containing up to 6 carbon atoms or an aryl or aralkyl group optionally carying an additional group W, or to each pair of adjacent groups R3 and R10 are annealed, with due regard for the interspersed C atoms, 5- to 6-member rings that may be saturated, unsaturated or aromatic, and that optionally carry an additional group R, and X and Y are the same or different, each independently representing an O, S, Se or Te or a -C(CH3)2-, -CH=CH- or -CR13R14- group, where R13 and R14 independently represent a hydrogen atom, a group B or W, or an alkyl or alkylene group containing up to 6 carbon atoms or an aryl or aralkyl group containing up to 9 carbon atoms, the alkyl, alkenyl, aryl or aralkyl group optionally carrying an additional group W, on the condition that, where X and Y are selected from -O-, -S-, -CH=CH-, and -C(CH3)2-, at least one of the residues R3 to R12 is a non-selectively bonding macromolecule or residue of formula VI
~(O)v~(CH2)o~CO~NR34~(CH2)s~(NH-CO)q~R35 (VI);
where o and s equal 0 or independently represent an integer between 1 and 6, q and v independently represent 0 or 1, R34 represents a hydrogen atom or a methyl residue, R35 represents an alkyl residue containing 3 to 6 C atoms and comprising 2 to n-1 hydroxy groups, with n being the number of C atoms, or an alkyl residue containing 1 to 6 C atoms that carries 1 to 3 additional carboxy groups, an aryl residue containing 6 to 9 C atoms or arylalkyl residue containing 7 to 15 C atoms, or a residue of the general formula IIId or IIIe on the condition that q is 1, 62a wherein B is a biological detecting unit having a molecular weight of up to 30,000 that bonds to specific cell populations or selectively to receptors, or accumulates in tissues or tumors, or generally stays in the blood, or is a macromolecule that bonds non-selectively and W is a hydrophilic group that improves water-solubility;
wherein the cyanine dye is coupled to a biological detecting unit B.